Prostatic Binding Protein (PBP) and keratin have been established as markers to identify differentiated, acinar epithelial and undifferentiated, possibly basal, epithelial cells in primary cell cultures from rat ventral prostate tissue. Immunocytochemistry revealed that both PBP- and keratin-containing cells appear in primary culture. The abundance of each depends on culture conditions. Soon after aggregates of prostate cells were placed in primary culture, PBP synthesis and secretion fell and DNA synthetic activity in the epithelial cells increased. The amount of [3H]-labeled polyamines synthesized from [3H]ornithine fell transiently and rose again at the peak of DNA synthesis. Ornithine decarboxylase (ODC) activity fell along with PBP synthesis as DNA synthesis and level of [3H]polyamines increased. The results support others that suggest ODC activity in prostate epithelial cells is a correlate of cell function rather than proliferation. However, prostate epithelial cells retain the capacity to synthesize significant levels of polyamines from [3H]ornithine even when ODC activity is low during the DNA synthetic phase. We have demonstrated that PBP binds 7,12-dimethylbenz[a]anthracene and benz[a]pyrene with an even higher affinity than steroids. A new medium has been developed which selectively supports rapid proliferation of prostate epithelial cells and suppresses fibroblast proliferation.